Genome‐wide association study of cell‐mediated immune response in chicken

Cell‐mediated immunity (CMI) causes the intracellular destruction of the antigen or elimination of the host cell to make animals resistant against exogenous antigens and cancers. In this study, a genome‐wide association study (GWAS) was carried out to identify genomic regions associated with CMI in chicken using chicken 60k high‐density single nucleotide polymorphism (SNP) array. Genomic relationships were taken into account to adjust for population structure. In order to account for multiple testing, chromosome‐wise false discovery rate was controlled at 5% and 10% levels. Moreover, a comparison of the power of fixed and mixed linear models based on genomic inflation factor was carried out. Mixed linear model (MLM) had better inflation rate, and therefore the results from MLM were used for subsequent analysis. Three significantly associated SNPs (FDR < 0.05) on chromosome 24 and linkage group E22C19W28_E50C23, and three suggestively associated SNPs (FDR < 0.1) on chromosome 1, 5 and 16 were identified. Pathway analysis showed that two biological pathways, which are related to immune response, were strongly associated with the candidate genes surrounding identified SNPs, and their influences were mostly on antigen processing and presentation, and cellular structure.     

镉对体外培养鸡骨髓基质细胞成骨分化的毒性作用

骨是镉毒性作用的主要靶器官之一,但其对鸡骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和成骨分化的毒性作用仍不清楚.本研究利用差速贴壁纯化法获得鸡BMSCs,加入不同浓度镉处理不同时间,采用CCK-8法检测细胞增殖,碱性磷酸酶(alkaline phosphatase,ALP)和茜素...     

诱导鸡胚精原干细胞向成骨细胞分化的研究

为了探讨体外培养的鸡胚精原干细胞(SSCs)的多向分化潜能,取孵化18~20 d的鸡胚睾丸,无菌获取SSCs,体外培养传代,通过地塞米松、β-甘油磷酸钠、维生素C诱导鸡胚SSCs向成骨细胞分化,钙结节Von Kossa s法、改良的钙钴法及免疫组化法进行成骨细胞鉴定。结果显示SSCs被诱导15~21 d后分化为成骨细胞,诱导形成率为75%~80%;诱导后的细胞Von Kossa s染色可见细胞间布满黑色颗粒,提示有矿化基质沉积;改良钙钴法碱性磷酸酶染色胞浆呈深棕色或深黑色,免疫组化法染色细胞呈阳性。因此可以得出在不同的诱导条件下,SSCs体外可被定向诱导分化为成骨细胞,证明其具有多向分化潜能。     

Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

Adipocytes suppress differentiation of muscle cells in a co‐culture system

The development of adipose tissue in skeletal muscle is important for improving meat quality. However, it is still unclear how adipocytes grow in the proximity of muscle fibers. We hypothesized that adipocytes would suppress muscle cell growth so as to grow dominantly within muscle. In this study, we investigated the effect of adipocytes on the differentiation of muscle cells in a co‐culture system. The fusion index of C2C12 myoblasts co‐cultured with 3T3‐L1 adipocytes was significantly lower than that of the control. The expression of myogenin and myosin heavy chain in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes was significantly lower than in the control. Furthermore, the expression of Atrogin‐1 and MuRF‐1 was higher in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes than the control. These results suggest that 3T3‐L1 adipocytes suppress the differentiation of C2C12 myoblasts. In addition, 3T3‐L1 adipocytes induced the expression and secretion of IL‐6 in C2C12 muscle cells. The fusion index and myotube diameter were higher in C2C12 muscle cells co‐cultured with 3T3‐L1 cells in medium containing IL‐6‐neutralizing antibody than the control. Taken together, there is a possibility that adipocyte‐induced IL‐6 expression in muscle cells could be involved in the inhibition of muscle cell differentiation via autocrine.     

Influences of protein ingestion on glucagon‐like peptide (GLP)‐1‐immunoreactive endocrine cells in the chicken ileum

Influences of a specific dietary nutrient on glucagon‐like peptide (GLP)‐1‐containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP‐1‐containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP‐1‐immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP‐1‐immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP‐1‐immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm², mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P < 0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP‐1‐immunoreactive cells. The protein ingestion is one of the signals that influence GLP‐1‐containing cells in the chicken small intestine.     

Influence of stewing time on the texture,ultrastructure and in vitro digestibility of meat from the yellow‐feathered chicken breed

This study assessed the influence of stewing (1, 2 or 3 h) on the texture, ultrastructure and in vitro digestibility of meat from the yellow‐feathered chicken, which is a popular broiler chicken in Asia. Results indicated that longer stewing considerably increased cooking loss of the chicken carcass and tenderness of thigh meat. After 3 h of stewing, protein digestibility decreased from 90.5% to 80.3% and fiber diameter decreased by 8.63 μm. The shear force value of breast meat decreased from 32.34 N at 1 h to 10.29 N at 2 h, and then increased to 39.98 N at 3 h. The texture profile of breast meat remarkably decreased during stewing. Moreover, increased stewing time induced longitudinal and transversal shrinkage of muscle fibers and the degradation of the myosin heavy chain. These findings suggested that prolonged stewing (3 h) resulted in decreased meat qualities, based on the changes in cooking loss, digestibility and texture profile, but that stewing for 2 h increased thigh and breast tenderness. Producers could utilize this information to stew yellow‐feathered chicken meat with desirable qualities.     

Comparative analysis of mechanical stretch-induced activation activity of back and leg muscle satellite cells in vitro

It has previously been shown that mechanical stretch induces activation of cultured quiescent satellite cells by rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c‐met receptor. The present study provides evidence that the stretch activation activity varies according to the origin of satellite cells from back and leg skeletal muscles in vitro. Satellite cells were isolated from three muscle groups, back (BK), upper hind limb (UL) and lower hind limb (LL) muscles, of adult male rats and stretch activation activities were compared. In response to stretch, lower hind limb satellite cells showed significantly greater response than upper hind limb and back muscles (LL > UL > BK). Immunoblots of stretched culture media revealed a higher HGF‐releasing capacity of lower hind limb satellite cells than back muscle satellite cells. In addition, lower hind limb satellite cells exhibited a greater activation activity in response to exogenous HGF added to culture media than compared to satellite cells from back and upper hind limb (LL > UL > BK). The increased ability to release HGF and the increased cellular responsiveness might account for higher stretch activation activities of lower hind limb satellite cells. Electrophoretic analysis of myosin heavy chain isoforms verified a higher content of slow muscle fibers in lower limb muscles (LL > UL > BK), suggesting a difference in stretch‐induced activation activity between satellite cells associated with fast and slow muscle fibers.     

Effect of dietary nutmeg oil on heat‐stress tolerance‐related parameters in Korean native chicken reared under hot temperature

This study investigated the effect of dietary nutmeg oil (NO) on growth performance, blood parameters, lipid peroxidation and heat shock protein (HSP) 70 expression in Korean native chicken (KNC) reared under hot temperature. We allocated 273 meat‐type KNCs (Hanhyup‐3, 4‐week‐old, body weight [BW] = 539.93 ± 1.75 g) to the following three treatments with seven replicate pens (13 birds/pen) per treatment. Three treatment diets were as follows: (a) Control, basal diet without NO supplementation; (b) NO 250; and (c) NO 500, basal diet supplemented with 250 and 500 ppm NO respectively. Diets and water were provided ad libitum throughout the 6‐week feeding trial. During overall period (0–6 weeks), no differences (p > 0.05) were observed in BW gain (BWG), feed intake (FI) and feed conversion rate (FCR) among treatments. However, the FI at 0–3 weeks decreased (p < 0.05) quadratically with increasing NO levels. Most blood parameters did not differ (p > 0.05) among treatments, although the monocyte level of the NO 500 group was considerably lower (p > 0.05) than that of the other groups. Furthermore, dietary NO did not affect serum triglyceride, cholesterol, total protein, albumin, calcium, phosphorus and alanine aminotransferase (ALT) levels (p > 0.05); however, it linearly decreased serum aspartate aminotransferase (AST) level (p < 0.05). Additionally, serum malondialdehyde (MDA) concentration decreased (p < 0.05) and heart MDA concentration was lower (p = 0.08) with increasing dietary NO supplementation. After a 3‐hr heat (35°C) challenge, the rectal temperature (RT) reduced (p < 0.05) linearly with increasing NO levels. Dietary NO did not affect liver HSP70 (p > 0.05) gene expression. In conclusion, NO potentially enhanced the ability of chickens to alleviate heat stress. Furthermore, our findings suggest that lipid oxidation inhibition by dietary NO likely mediated the enhanced heat‐stress tolerance of the chickens.     

In vitro measurement of post‐natal changes in proliferating satellite cell frequency during rat muscle growth

Satellite cells, resident myogenic stem cells found in postnatal skeletal muscle, are most abundant during early postnatal development and sharply decline in frequency thereafter to adult levels in mice and rats. Therefore, postnatal changes in satellite cell mitotic activities are important aspects for further understanding a muscle growth strategy. In large meat‐production animals, however, the traditional in vivo proliferation assay may be less realistic because it requires intra‐peritoneal (ip) injection of huge dosage of mutagenic nucleosides, ³H‐labeled thymidine or bromodeoxyuridine (BrdU), at each age‐time of sacrifice. We report in the present pilot study using rats that in vivo proliferation activity of satellite cells can be evaluated by an in vitro BrdU‐incorporation assay in early cultures. Briefly, satellite cells were prepared from upper hind‐limb and back muscles and maintained for 24 h with imposing by BrdU addition for the last 2 h, followed by the regular immunocytochemistry for determining BrdU‐incorporated cell percentage. This in vitro assay demonstrated a rapid decrease in proliferating satellite cell frequency to the adult level during about 3‐month period after birth, and yielded a high correlation to the measurements by the in vivo BrdU ip‐injection method during the postnatal period examined from day‐2 to month‐11. The in vitro proliferation assay may be further adaptable for large domestic animals by the combination with a muscle biopsy technique that enables age‐interval sampling from the same growing animals.     

Involvement of μ/m‐calpain in the proteolysis and meat quality changes during postmortem storage of chicken breast muscle

The objective of this study was to investigate the role of calpain isotypes, especially poultry‐specific μ/m‐calpain in the proteolysis and meat quality changes of chicken breast muscle during postmortem storage. Calpain activity was detected by casein zymography, while the degradation of titin, desmin and Troponin‐T was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and western blot. Meat quality indicators such as water holding capacity and tenderness were also studied. The correlation analysis between calpain activity, proteolysis and the changes in meat quality indicators indicated that there were strong correlations for μ‐calpain during the first 12 h of storage, while such strong correlations for μ/m‐calpain were only found in samples stored from 12 h to 7 days. Our study suggested that μ‐calpain played a major role in meat quality changes while μ/m‐calpain could also be involved but played a limited role in the proteolysis and meat quality changes during 12 h to 7 days postmortem storage of chicken breast muscle.     

Effects of (−)‐hydroxycitric acid on lipid droplet accumulation in chicken embryos

This study was conducted to determine the impact of (?)‐hydroxycitric acid ((?)‐HCA) on biochemical indices and lipid metabolism parameters in chicken embryos. Two hundred and forty fertilized eggs were divided into six groups and injected with (?)‐HCA at concentrations of 0, 0.1, 0.5, 1.0, 10.0 and 50 mg/kg (n = 40). After 19 days of incubation, serum and liver were collected for analysis of biochemical indices and lipid metabolism parameters. Results showed no significant differences on serum biochemical indices: 1–50 mg/kg (?)‐HCA significantly increased serum glucose and hepatic glycogen contents (P < 0.05). Oil Red O staining analysis showed total area, counts of lipid droplets and hepatic triglyceride content were significantly decreased (P < 0.01), meanwhile hepatic lipase and lipoprotein lipase activity were significantly increased (P < 0.05). ACLY, ME1, SREBP‐1c messenger RNA (mRNA) levels in 0.5–10 mg/kg groups and FAS mRNA level in 1–10 mg/kg groups were significantly decreased (P < 0.05), while PPARα mRNA level, serum adiponectin content and AdipoR1 mRNA level were significantly increased in 0.5–50 mg/kg groups (P < 0.05). These results indicated (?)‐HCA treatment inhibited triglyceride synthesis via decreasing lipogenesis‐related factors, mRNA expression level and accelerated lipolysis by enhancing lipoprotein lipase and hepatic lipase activity, which finally reduced lipid droplet accumulation, and this action may be associated with activating the adiponectin signaling pathway.     

狗舌草提取物诱导淋巴细胞性白血病L1210细胞分化的研究

以单猪屎豆碱（MO）、槲皮素（QU）和环磷酰胺（CY）为参照，观察了狗舌草600mL／L乙醇提取物（EX）对淋巴细胞性白血病L1210细胞体外试验的形态变化；利用流式细胞术，从DNA分子水平上检查了EX对L1210细胞各周期相的影响，探讨EX对L1210细胞的分化机理。结果发现，EX能够使L1210细胞向淋巴细胞方向发展；经EX作用24h后，L1210细胞G0＋G1期的百分比较对照组明显升高。提示EX对L1210细胞增殖的抑制作用可能是由于G1期的阻滞所致。     

Mechanical stretch-induced activation of skeletal muscle satellite cells is dependent on nitric oxide production in vitro

Mechanical stretch induces activation of cultured quiescent satellite cells and the activation response is owing to rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. We provide new evidence that the stretch activation is dependent on nitric oxide (NO) production. Stretch activation could be abolished by the addition of N ^G-nitro- L -arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, but not by N ^G-nitro- D -arginine methyl ester hydrochloride, a less active enantiomer of L-NAME. Adding HGF to the L-NAME culture restored the activation response, indicating that L-NAME does not directly inhibit satellite cell activation, but acts upstream from the HGF release. In addition, immunoblots of satellite cell lysate revealed the presence of nitric oxide synthase. These experiments suggest that NO is involved in linking mechanical perturbation of satellite cells to chemical signaling responsible for HGF release from its sequestration in vitro .     

Potential role of ALDH3A2 on the lipid and glucose metabolism regulated by (‐)‐hydroxycitric acid in chicken embryos

This study aimed to investigate the effect of (‐)‐hydroxycitric acid ((‐)‐HCA) on lipid and glucose metabolism, and further analyzed these actions whether associated with modulation of aldehyde dehydrogenase 3 family member A2 (ALDH3A2) expression in chicken embryos. Results showed that (‐)‐HCA decreased triglyceride content and lipid droplet counts, while these effects induced by (‐)‐HCA were reversed in chicken embryos pre‐transfected with sh4‐ALDH3A2. (‐)‐HCA decreased malic enzyme, acetyl‐CoA carboxylase, fatty acid synthase, and sterol regulatory element binding protein‐1c mRNA level, while increased carnitine palmitoyl transferase 1A (CPT1A) and peroxisome proliferators‐activated receptor α (PPARα) mRNA level; and the action of (‐)‐HCA on lipid metabolism factors had completely eliminated in embryos pre‐transfected with sh4‐ALDH3A2. Chicken embryos pre‐transfected with sh4‐ALDH3A2 had eliminated the increasing of serum glucose and hepatic glycogen content induced by (‐)‐HCA. (‐)‐HCA decreased phosphofructokinase‐1 and increased G6P, fructose‐1,6‐bisphosphatase, phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate carboxylase mRNA level in chicken embryos. Similarly, the effect of (‐)‐HCA on these key enzyme mRNA level was reversed in embryos pre‐transfected with sh4‐ALDH3A2. Furthermore, (‐)‐HCA increased PPAR‐γ‐coactivator‐1α (PGC‐1α), PPARα, hepatic nuclear factor‐4A, PEPCK, and CPT1A protein level, and these actions of (‐)‐HCA disappeared in embryos pre‐transfected with sh4‐ALDH3A2. These results indicated that (‐)‐HCA reduced fat accumulation and accelerated gluconeogenesis via activation of PGC‐1α signaling pathway, and these effects of (‐)‐HCA might associate with the increasing of ALDH3A2 expression level in chicken embryos.     

The effect of feed supplementation with a copper‐glycine chelate and copper sulphate on selected humoral and cell‐mediated immune parameters,plasma superoxide dismutase activity,ceruloplasmin and cytokine concentration in broiler chickens

The varied bioavailability and different effects of organic forms of copper on the immune system of poultry have prompted the search for new feed additives based on copper compounds containing modified chelate complexes. The aim of the study was to determine the effect of inorganic and organic forms of copper on selected parameters of the cellular and humoral immune response in broiler chickens by determining the percentages of CD3⁺CD4⁺, CD3⁺CD8⁺ and CD25⁺ lymphocytes, cells with MHC Class II expression, and BU‐1⁺ cells, as well as the concentrations of SOD, IL‐2, IL‐10 and TNF‐α in the peripheral blood. The experiments were conducted using 500 one‐day‐old Ross 308 roosters divided into five groups. Cu was added in inorganic form (CuSO₄), in inorganic form with the addition of phytase (CuSO4 + F), in organic form in combination with glycine (Cu‐Gly) and in organic form in combination with glycine and a phytase supplement (Cu‐Gly+F). The results of the study indicate an increase in the percentage of CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes, CD25⁺ T cells, and cells expressing MHC class II molecules, and in the concentration of ceruloplasmin, activity of superoxide dismutase and the concentration of IL‐2 in the groups that received copper, particularly copper‐glycine chelates. Based on the study, we can conclude that supplementation of poultry feed with copper chelates activates mainly the Th1 cellular immune response and the response of peripheral blood T lymphocytes. Furthermore, it promotes secretion of cytokines, which are involved in potentiation and regulation of the immune response in birds.     

Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro

The present study evaluates the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chicken enterocyte and to establish an effective technique for chicken intestinal epithelial cell (IEC) cultivation. Fourteen‐day‐old, 16‐day‐old and 18‐day‐old embryos (average weight: 52.23 ± 0.76 g, 50.86 ± 0.99 g, 48.98 ± 1.03 g) were the source for preparation of enterocyte culture, and trypsin‐ethylene diamine tetraacetic acid, collagenase, thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determined by qualitative assays of proliferation. Cells isolated by using 14‐day‐old embryo and collagenase obtain the best attachment and growth in culture, and the production of continuously growing IEC cultures. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14‐day‐old embryo as a source can be advantageously applied to the isolation of chicken IEC and this method may be useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.     

Effects of the estrous cycle on the efficacy of oocyte collection and in vitro embryo production in Duroc‐breed

Collection efficacy and in vitro embryo developmental ability of oocytes obtained from Duroc‐breed ovary donors at different stages of the estrous cycle (days 6, 12 and 16 after estrus) were performed. The numbers of collected oocytes did not differ significantly among the different estrous cycle groups (total 72–90 oocytes per gilt). However, the blastocyst rates of oocytes collected on days 12 and 16 (9.2% and 19.4%, respectively) were significantly higher than those on day 6 (1.1%). More oocytes were obtained on day 16 from small follicles (<2 mm in diameter; 85.3 oocytes per gilt) than from medium‐sized (≥2–<6 mm) and large (≥6 mm) follicles (17.5 and 12.8 oocytes, respectively). The blastocyst rates in both the medium‐sized and large follicle groups (20.0% and 19.2%, respectively) were significantly higher than that in the small follicle group (6.3%). The blastocyst cell numbers in both the medium‐sized and large follicle groups (39.4 and 43.3 cells, respectively) were significantly higher than that in the small follicle group (30.6 cells). The results suggest that oocyte collection from cycling Duroc pigs can be carried out efficiently from the late luteal to follicular stage. Those oocytes collected from medium‐sized and large follicles show better embryo development.     

The effects of environmental enrichment and transport stress on the weights of lymphoid organs,cell‐mediated immune response,heterophil functions and antibody production in laying hens

The effects of environmental enrichment and transport stress on the immune system were investigated in laying hens. A total of 48 1‐day‐old chickens were used, half of the chickens were reared in conventional cages (RCC) and the rest in enriched cages (REC). Transport stress was applied in the 17th week. Liver weight decreased, spleen and bursa of Fabricius weights, white blood cell count, CD4+ and CD8+ cell proportions increased due to the transport. Environmental enrichment significantly increased antibody production and tended to increase monocyte percentage and CD8+ cell proportion. The effect of transport on, heterophil (H) and lymphocyte (L) percentages was not significant in RCC chickens. While heterophil percentage and H:L ratio increased, lymphocyte percentage decreased in REC chickens subjected to transport. Transport stress increased heterophil functions both in REC and RCC chickens, but the increase was higher in REC hens than in RCC hens. In conclusion, although environmental enrichment did not neutralize the effect of transport on lymphoid organs, it activated the non‐specific immune system, cellular and the humoral branches of the specific immune system by increasing heterophil functions, CD8+ cells and antibody production, respectively. Therefore, environmental enrichment suggested for improving animal welfare may also be beneficial to improve the immune system of birds exposed to stress.